Intracellular levels of calcium are regulated by movement of calcium through channels in cellular membranes. Calcium release through an ion channel present in the terminal cisternae of the sarcoplasmic reticulum initiates contraction in skeletal muscle. This Ca.sup.2+ release channel has been shown to bind the neutral plant alkaloid ryanodine with high affinity and can be regulated by ryanodine, Ca.sup.2+, ATP, KCl, Mg.sup.2+ ruthenium red, caffeine and calmodulin.
The use of radiolabeled ryanodine as a probe has led to the identification, purification and biochemical characterization of the Ca.sup.2+ release channel/ryanodine receptor from skeletal, cardiac and neuronal tissue. The predicted primary structure of the Ca.sup.2+ release channel/ryanodine receptor has been determined in several species and tissues (see e.g., Takeshima et al., Nature 339, 439-445 (1989); Zorzato et al., J. Biol. Chem. 265, 2244-2256 (1990); and Fujii et al., Science 253, 448-451 (1991)), but the specific site modulated by ryanodine is unknown.
Ryanodine has different effects on the channel conductance and gating properties depending on the type of tissue and concentration of ryanodine. In skeletal and cardiac muscle for instance, micromolar concentrations of ryanodine inhibit Ca.sup.2+ release by closing the Ca.sup.2+ release channel, while nanomolar concentrations of ryanodine stimulate Ca.sup.2+ release by locking this channel in the open state. In neuronal tissue, micromolar concentrations of ryanodine completely block the channel, however, lower concentrations of ryanodine do not appear to affect the brain Ca.sup.2+ release channel.
Knowledge about the site of action for ryanodine could provide additional information about receptor structure and function. Since immunologic probes have been widely used for the characterization of membrane proteins and ion channels, one approach toward characterization of the ryanodine binding site involves the production of high-affinity antibodies to ryanodine which mimic the binding characteristics of the skeletal muscle membrane receptor.
Preparation of antibodies to peptides or small molecules like ryanodine (M.W. 493) is usually accomplished by conjugation to a carrier protein for optimal antigenicity. However, derivatization of ryanodine to produce an immunogenic conjugate which retains the essential ryanodine binding properties has proven to be extremely difficult.